Elsevier

Urology

Volume 82, Issue 6, December 2013, Pages 1453.e1-1453.e7
Urology

Basic and Translational Science
Gene Expression Profile During Testicular Development in Patients With SRY-negative 46,XX Testicular Disorder of Sex Development

https://doi.org/10.1016/j.urology.2013.08.040Get rights and content

Objective

To elucidate alternative pathways in testicular development, we attempted to clarify the genetic characteristics of SRY-negative XX testes.

Materials and Methods

We previously reported 5 cases of SRY-negative 46,XX testicular disorders of sex development and demonstrated that coordinated expression of genes such as SOX9, SOX3, and DAX1 was associated with testicular development. We performed a case-control study between the aforementioned boy with 46,XX testicular disorders of sex development and an age-matched patient with hydrocele testis (46,XY). During their consecutive surgeries, testicular biopsy specimens were obtained. Genes with differential expression compared with XY testis were identified using polymerase chain reaction (PCR)–based subtractive hybridization and sequencing. For validation of differential gene expression, real-time RT-PCR was performed using gene-specific primers. The distribution of candidate proteins in the testicular tissue was clarified by immunohistochemistry in human and rodent specimens. Moreover, in vitro inhibitory assays were performed.

Results

We identified 13 upregulated and 7 downregulated genes in XX testis. Among the candidate genes, we focused on ROCK1 (Rho-associated, coiled-coil protein kinase 1) in the upregulated gene group, because high expression in XX testis was validated by real-time RT-PCR. ROCK1 protein was detected in germ cells, Leydig cells, and Sertoli cells by immunohistochemistry. Moreover, the addition of specific ROCK1 inhibitor to Sertoli cells decreased SOX9 gene expression.

Conclusion

On the basis of in vitro inhibitory assay, it is suggested that ROCK1 phosphorylates and activates SOX9 in Sertoli cells. Testes formation might be initiated by an alternative signaling pathway attributed to ROCK1, not SRY, activation in XX testes.

Section snippets

Ethics Statement

Studies using human testicular tissue were carried out after obtaining written informed consent from the families of the patients and the approval of the Institutional Review Board of Nagoya City University Hospital (approval number 83). All animal experimental procedures were performed in accordance with protocols approved by the Animal Care Committee of Nagoya City University Graduate School of Medical Sciences (protocol number: H23M-20).

Patients and Sample Preparation

We had previously examined 5 patients with 46,XX

Results

The patient with 46,XX testicular DSD analyzed herein presented with hypospadias and bilateral cryptorchidism, and multiple surgeries were performed. Peripheral blood sample analysis revealed no expression of SRY or DAZ genes in this patient using by fluorescence in situ hybridization methods. We further investigated the testicular tissue of this patient and age-matched XY tissue derived from the patient with hydrocele. Histologic findings of XX and XY testes are shown in Figure 1A, B. Both the

Comment

The mechanisms mediating testicular development in SRY-negative 46,XX testicular DSD patients, such as the patient presented in this study, remain unclear. In the present study, we investigated the differential expression of genes between XX and XY testes using PCR-based subtractive hybridization and detected 13 upregulated genes in XX testes. In this study, we confirmed the overexpression of the ROCK1 gene and found that the expression of ROCK1 protein was increased in Sertoli cells of XX

Conclusion

We identified ROCK1 as the differentially expressed gene between XX and XY testes. ROCK1 protein was detected in the germ cells and Sertoli cells. On the basis of the results of in vitro inhibition assay, we hypothesized that ROCK1 phosphorylates and activates SOX9 in Sertoli cells. It is suggested that testis formation is initiated by an alternative signaling pathway attributed to ROCK1, and not SRY, activation in 46,XX testicular DSD.

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    Financial Disclosure: The authors declare that they have no relevant financial interests.

    Funding Support: This article was supported by funding from a Grant-in-Aid for Scientific Research (B) of Japan Society for the Promotion of Science (40238134).

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